Clinical validation of a next-generation sequencing assay specifically for blood-drop liquid biopsy

Chen-Hsiung Yeh1, Jonathan Spurgin1, Andrew Ford1, John Athanasuleas1, Upender Manne2

1Circulogene Theranostics, Homewood, AL; 2Department of Pathology, University of Alabama at Birmingham, Birmingham, AL

RESULTSABSTRACT RESULTS (Continued)

1. Ford A, Spurgin J, Athanasuleas J & Yeh CH (2015) J Cancer Prev Curr Res 3(1): 00064 DOI: 10.15406/jcpcr.2015.03.00064

2. Spurgin J, Ford A, Athanasuleas J, Yeh CH (2015) Intl J Life Sci Res Vol. 3, Issue 3, pp: 23-29.

3. Yeh CH (2015) MOJ Cell Science & Report. Volume 2, Issue 3.4. McLarty, JL, Yeh CH (2015) MOJ Cell Science & Report. Volume 2, Issue 2.5. Yeh CH, Spurgin J, Ford A, Athanasuleas J, McLarty J, Mullen M (2015) J Clin Oncol 33

(Suppl: abstr e22008). 2015 ASCO Annual Meeting.

The liquid biopsy industry is utilizing an extraction methodflawed for clinical application. Clinical validation ofCirculogene’s blood-drop liquid biopsy NGS will provide afirst-in-class offering – matching each patient’s cancermutations with specifically targeted therapy and follow-up.

Next-generation sequencing (NGS) technology enables rapidanalysis and turnaround of multiple genes for clinicallyactionable somatic variants. A liquid biopsy, based oncirculating cell-free DNA (cfDNA), can capture the entireheterogeneity of the disease, and offer what tissue biopsiescan’t, the opportunity to take serial samples in order tomonitor tumor genomic evolution in real time. We haverecently developed a proprietary cfDNA enrichment processthat requires only droplet volumes of blood. Here, wepresented clinical validation of the blood-drop liquid biopsyNGS assay interrogating 2855 mutation hotspots in 50cancer-associated genes using the AmpliSeq cancer paneland Ion Torrent Proton sequencer.

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Our new-generation liquid biopsy NGS assay, with thecapability to multiplex and simultaneously sequence multiplepatient samples using finger-stick blood, is a robust andsensitive method for mutation analysis of clinical significance,further expedite treatment decision-making and identifytargeted therapies for eligible patients in a time- and cost-efficient manner.

• CLIA-certified

• Minimal input with maximal output

• NGS-grade cfDNA quality• Most time- and cost-efficient

1. NGS Quality Controls Used in This Validation (Ave. Coverage >200X)

OBJECTIVESCONCLUSIONS

REFERENCES

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METHODSBlood was collected in EDTA-containing tubes, centrifuged,and circulating cfDNA was recovered from 20 uL of plasma byCirculogene’s proprietary cfDNA enrichment technology.Targeted sequencing was performed using Ion AmpliSeqCancer Hotspot Panel v2 on Ion Proton with 1 ngcfDNA input.Sequencing data were analyzed by Variant Caller 4.0 andGenePool software (Station X).

2. Test Sensitivity Down to 0.5-1% 4. Test Specificity

3. Test Reproducibility and Repeatability

5. Test Accuracy (Inter-Lab Correlation)

Sample ID CGT Atherotech ConcordanceWT1 PDG FRA ( CO SM 22413) ,

TP53 ( CO SM 43606, CO SM 39293,CO SM 179807, CO SM 179806,CO SM 179805, CO SM 44683)

PDG FRA ( CO SM 22413) ,TP53 ( CO SM 43606, CO SM 39293,

CO SM 179807, CO SM 179806,CO SM 179805, CO SM 44683)

100%

WT2 PI K3CA ( CO SM 758) , APC ( CO SM 19099)

PI K3CA ( CO SM 758) , APC ( CO SM 19099)

100%

WT3 PDG FRA ( CO SM 22413) ,M ET ( NO CO SM 988) ,BRAF ( CO SM 1116) ,RET ( CO SM 29804) ,PTEN ( CO SM 5101) ,ATM ( CO SM 21825) ,TP53 ( CO SM 12296) ,

SM ARCB1 ( CO SM 1090)

PDG FRA ( CO SM 22413) ,M ET ( NO CO SM 988) ,BRAF ( CO SM 1116) ,RET ( CO SM 29804) ,PTEN ( CO SM 5101) ,ATM ( CO SM 21825) ,TP53 ( CO SM 12296) ,

SM ARCB1 ( CO SM 1090)

100%

WT4 PDG FRA ( CO SM 22413) ,NRAS ( CO SM 587) ,VHL ( CO SM 14355) ,

PI K3CA ( CO SM 94986, CO SM 775) ,BRAF ( CO SM 1116) ,PTEN ( CO SM 5111)

PDG FRA ( CO SM 22413) ,NRAS ( CO SM 587) ,VHL ( CO SM 14355) ,

PI K3CA ( CO SM 94986, CO SM 775) ,BRAF ( CO SM 1116) ,PTEN ( CO SM 5111)

100%

WT5 PI K3CA ( CO SM 14052) ,KI T ( CO SM 21983) ,APC ( CO SM 19099) ,BRAF ( CO SM 1116) ,

G NAQ ( CO SM 28760) ,TP53 ( CO SM 45329, CO SM 43960,

CO SM 46214)

PI K3CA ( CO SM 14052) ,KI T ( CO SM 21983) ,APC ( CO SM 19099) ,BRAF ( CO SM 1116) ,

G NAQ ( CO SM 28760) ,TP53 ( CO SM 45329, CO SM 43960,

CO SM 46214)

100%

WT6 TP53 ( CO SM 11517) TP53 ( CO SM 11517) 100%

WT7 PDG FRA ( CO SM 22413) ,KI T ( CO SM 1290) ,

APC ( CO SM 19049) ,TP53 ( CO SM 44512, CO SM 45511)

PDG FRA ( CO SM 22413) ,KI T ( CO SM 1290) ,

APC ( CO SM 19049) ,TP53 ( CO SM 44512, CO SM 45511)

100%

WT8 STK11 ( CO SM 25851) STK11 ( CO SM 25851) 100%

WT9 KI T ( CO SM 28026) KI T ( CO SM 28026) 100%

WT10 APC ( CO SM 13125) ,TP53 ( CO SM 44973) ,ERBB2 ( CO SM 35496)

APC ( CO SM 13125) ,TP53 ( CO SM 44973) ,ERBB2 ( CO SM 35496)

100%

WT11 BRAF ( CO SM 21542) ,ATM ( CO SM 21826) ,TP53 ( CO SM 45169)

BRAF ( CO SM 21542) ,ATM ( CO SM 21826)

One var iant was not called by At her ot ech’s assay

WT12 EG FR ( CO SM 41603, CO SM 41663),M ET ( CO SM 710) ,BRAF ( CO SM 461)

EG FR ( CO SM 41603, CO SM 41663),M ET ( CO SM 710) ,BRAF ( CO SM 461)

100%

WT13 M ET ( CO SM 691) ,TP53 ( CO SM 10995)

M ET ( CO SM 691) ,TP53 ( CO SM 10995)

100%

WT14 ATM ( CO SM 21626) ,TP53 ( CO SM 43879) ,

SM AD4 ( CO SM 13115)

ATM ( CO SM 21626) ,TP53 ( CO SM 43879) ,

SM AD4 ( CO SM 13115)

100%

WT15 PDG FRA ( CO SM 22413) ,M ET ( NO CO SM 988) ,BRAF ( CO SM 1116) ,

TP53 ( CO SM 11738, CO SM 44848)

PDG FRA ( CO SM 22413) ,M ET ( NO CO SM 988) ,BRAF ( CO SM 1116) ,

TP53 ( CO SM 11738, CO SM 44848)

100%

WT16 No m ut at ion det ect ed No m ut at ion det ect ed 100%

WT17 VHL ( CO SM 30295) ,ERBB2 ( CO SM 35496)

VHL ( CO SM 30295) ,ERBB2 ( CO SM 35496)

100%

Shown in Table 4 are the variant calls of genes and mutations indicated byCOSMIC ID numbers, silent mutations and unconfirmed somatic mutationsare NOT filtered out. Different COSMIC ID may represent the samemutation.

The read depth for each sample is at least 2500X. Tested samples include17 apparently normal healthy subjects.

Test specificity is 99.9% at gene level (849/850) and in strong concordance(94%; 16/17) with another CLIA laboratory

COSMIC IDs were taken from the Catalogue of Somatic Mutations inCancer: http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/

*Shown in t his t able ar e t hevar iant calls of genes andm utat ionsindicat ed byCO SM IC I D num ber s, silent m utat ionsand unconfir m edsom at ic m ut at ions ar eNO Tf ilt er edout . Dif fer ent CO SMI C ID may repr esent the same mut ation. Ther eaddept hf or eachsample isbet ween 1100X – 5700X. Test ed sam ples include 15pancr eatic, 15color ectal, 1G IST, 4lung, 15 CEA- posit ive serum sam ples( samplenum ber s wit h “ S” ) .